Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

Summary

This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction.

Abstract

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with muscular dystrophy is an invaluable cellular source for in vitro disease model systems and can be used for drug screening studies. Patient-derived urine cells have been used in successful reprogramming into induced pluripotent stem cells in order to model dystrophic cardiomyopathy¹. Addressing the safety concerns of integrating vector systems, we present a protocol using a non-integrating Sendai virus vector for transduction of Yamanaka factors into urine cells collected from patients with muscular dystrophy. This protocol generates fully reprogrammed clones within 2–3 weeks. The pluripotent cells are vector-free by passage-13. These dystrophic iPSCs can be differentiated into cardiomyocytes and used either to study disease mechanisms or for drug screening.

Introduction

Cardiomyopathy is the second leading cause of death in patients with Duchenne and Becker muscular dystrophy (MD). Although mutations in the X-linked dystrophin gene occur in 1:3,500 male births, very little is known about the molecular and cellular events leading to progressive cardiac muscle damage. Human induced pluripotent stem cells derived from muscular dystrophy patients have emerged as a novel tool to study the underlying disease mechanisms and to use for drug screening^1,2.

The anticipated discomfort of skin biopsies or blood samples may dissuade young patients and/or their guardians to give consent for study participation....

Protocol

NOTE: Patients and/or their guardians should give informed consent to participate in an Institutional Review Board approved study.

1. Buffers and Media Preparations

1. Washing buffer: To prepare 100 ml of washing buffer, add 1 ml of 100x pen/strep Solution (100 U/ml penicillin + 100 µg/ml of Streptomycin) to 99 ml of phosphate buffer saline (PBS).
2. Urinary Progenitor Cell (UPC) Medium: To prepare UPC medium, mix equal volumes of Keratinocyte Serum Free (KSF) Medium + Progenitor Cell Medium.
   1. KSF Medium: To prepare keratinocyte medium, add 5 ng/ml of epidermal growth factor (EGF), 50 ng/ml of bovine pituitar....

Results

Most progenitor cells isolated from human urine are positive for uroepithelial progenitor and pericyte markers such as CD44, CD73, and CD146 (97.37%, 97.09%, and 97.3% respectively; Figure 1A and 1B). These cells also expressed other mesenchymal markers such as alpha-smooth muscle actin and vimentin (Figure 1B). RT-PCR analysis gives evidence of a mixed population of cells in the cultures in that there is weak expression of Cytokeratin-7 (CK-7) and Uroplakin (UP)-Ia &.......

Discussion

Modeling cardiovascular diseases using iPSCs is becoming a common approach to understand the genetic contribution^4-6. Some unanticipated difficulties of obtaining cell samples from patients, especially young children, can be avoided by offering the option of a non-invasive approach such as a urine collection. In this young patient population, it is often difficult to collect a volume of urine sufficient to yield enough urine cells for reprogramming. Coaching the young patients to drink fluids 30 min prior to t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Materials
4 Oz. Specimen Cup with Lid	STL Medical Supply	M9AMSAS340	For Urine Sample Collection
15 ml BD-Falcon Tubes	Fisher Scientific	352097
50 ml BD-Falcon Tubes	Fisher Scientific	352098
Round Glass Coverslips	Fisher Scientific	12-545-81
CytoTune-iPS Sendai Reprogramming Kit	Life Technologies	A1378-001
PBS, pH 7.4	Life Technologies	10010-023
Pen-Strep w/o Glutamine	Life Technologies	15140-122	Warm in 37 °C water bath before use
RPMI Medium 1640	Life Technologies	11875-093	Warm in 37 °C water bath before use
B-27 Supplement w/Insulin (50x)	Life Technologies	17504-044	Warm in 37 °C water bath before use
B-27 Supplement w/o Insulin (50x)	Life Technologies	0050129SA	Warm in 37 °C water bath before use
DMEM/F12 (1:1)	Life Technologies	11330-032	Warm in 37 °C water bath before use
Versene, 1:5,000	Life Technologies	15040066	Warm in 37 °C water bath before use
bFGF (10 µg)	Life Technologies	13256-029	Warm in 37 °C water bath before use
Cell Counter Cartridges	Life Technologies	C10228
Knockout Serum (500 ml Bottle)	Life Technologies	10828-028	Warm in 37 °C water bath before use
Recovery Cell Culture Freezing Medium	Life Technologies	12648-010
Keratinocyte-SFM (1x), Liquid	Life Technologies	17005-042	Warm in 37 °C water bath before use
DMEM, High Glucose, Glutamax	Life Technologies	10566-016	Warm in 37 °C water bath before use
Ham's F-12 Nutrient Mix	Life Technologies	11765-054	Warm in 37 °C water bath before use
EGF Recombinant Human Protein, Liquid Form	Life Technologies	PHG0311L	Warm in 37 °C water bath before use
Insulin, Human Recombinant, Zinc Soln	Life Technologies	12585-014	Warm in 37 °C water bath before use
TeSR-E8	Stem Cell Technologies	5940	Warm in 37 °C water bath before use
ROCK Inhibitor (Y-27632)	Selleck	S1049	Warm in 37 °C water bath before use
Matrigel	BD Biosciences	354277	hESC Qualified Matrix, LVED Free
RNeasy Mini Kit	Qiagen	74104
iScript cDNA Synthesis Kit	Bio-Rad	170-8890
DreamTaq Green PCR Master Mix (2x)	Thermo-Scientific	K1081
FBS-Qualified	Sigma Aldrich	F6178	Warm in 37 °C water bath before use
Adenine Bioreagent	Sigma Aldrich	A2786-5G	Warm in 37 °C water bath before use
Cholera Toxin from Vibrio cholerae	Sigma Aldrich	C8052-.5MG	Warm in 37 °C water bath before use
Hydrocortisone	Sigma Aldrich	H0888-1G	Warm in 37 °C water bath before use
Holo-transferrin from human	Sigma Aldrich	T0665-50MG	Warm in 37 °C water bath before use
3,3',5-Triiodo-L-thyronine sodium salt	Sigma Aldrich	T6397-100MG	Warm in 37 °C water bath before use
DAPI	Santa Cruz Biotechnology	SC-3598
Fluoromount Aquous mounting medium	Sigma Aldrich	F4680
16% Paraformaldehyde	Alfa-Aesar	43368
Antibodies
Mouse anti CD44 - labeled with FITC	BD Biosciences	560977
Mouse anti CD146 - labeled with PE	BD Biosciences	561013
Mouse anti CD73 - labeled with PE	BD Biosciences	561014
Mouse anti-α-smooth muscle actin	Sigma Aldrich	A2547
Mouse anti-Vimentin	Abcam	ab8978-100
Mouse Anti - TRA-1-81	Life Technologies	411100
Rabbit anti Oct 3/4	Santa Cruz Biotechnology	SC-9081
Mouse Anti Dystrophin	Leica Biosystems	NCL-DYSB